In our continuing effort to study the structure and function of polyoma virus T antigens, we have found that middle-size T antigen-associated protein kinase can exist as an active or inactive tyrosine-specific protein kinase. The active kinase is present exclusively in the plasma membrane, and appears to be phosphorylated in vivo at serine residues. These results suggest that the activity of "middle T kinase" may be regulated by a cellular plasma membrane enzyme. Antiserum against a synthetic polypeptide representing the region of middle-size T antigen believed to contain the phosphate acceptor acceptor site for the in vitro kinase reaction reacts exclusively with middle-size T antigen and blocks its in vitro phosphorylation. Polyoma virus mutants capable of growing in embryonic cells which are resistant to wild-type polyoma virus infection have also been isolated. In each case, a mutation was localized to the portion of the genome which regulates replication and DNA transcription. The ability of these virus mutants to grow in embryonic cells was found to depend on the stage of cellular differentiation.